Mitotic entry is driven by the explosive, autoregulatory activation of the kinase MPF (M phase promoting factor; Cdk1/cyclin B), which phosphorylates S/TP sites in a variety of mitotic phosphoproteins. Our laboratory has identified a novel kinase called Greatwall that is also required for the G2/M transition, in part by influencing the autoregulatory loop that activates MPF. However, Greatwall must also have other critical roles in the cell cycle. This conclusion arises from the fact that immunodepletion of Greatwall from Xenopus M phase-arrested CSF egg extracts causes a form of M phase exit, while in contrast, MPF is largely dispensable for maintaining M phase once the CSF state has been established. Our recent results indicate that: (1) MPF phosphorylates and helps activate Greatwall during M phase, and (2) Once activated, Greatwall function leads to the inactivation of a particular form of the phosphatase PP2A associated with the regulatory subunit B55delta. In this way, Greatwall protects the phosphorylations added by MPF to the S/TP sites on MPF substrates, including components of the autoregulatory loop. In the absence of Greatwall, PP2A/B55delta would immediately remove these phosphorylations from mitotic phosphoproteins; as a result, cells or extracts depleted for Greatwall in interphase cannot enter M phase, while cells or extracts depleted for Greatwall during M phase rapidly exit this state to interphase. In the first specific aim, we propose to dissect the pathway leading from Greatwall activation to PP2A/B55delta inactivation. To achieve this goal, we will identify the relevant substrate(s) phosphorylated by Greatwall, and we will also define the biochemical changes at the end of the pathway that block function of the phosphatase. In the second specific aim, we will place the pathway from Greatwall to PP2A/B55delta in the larger context of the cell cycle transitions that allow entry into, and exit from, M phase. We will identify the critical phosphorylations that activate Greatwall during M phase, and determine whether any of these are added to Greatwall by kinases other than MPF. One goal of this line of investigation is the generation of a constitutively active Greatwall protein that can be expressed in bacterial cells and used for studies of Greatwall structure. We will next determine how the activating phosphorylations on Greatwall, as well as the phosphorylations Greatwall adds to its substrates, are removed upon M phase exit. Finally, we will examine the rates of dephosphorylation in extracts of a large panel of phosphosites. In this way, we hope to find the rules governing the substrate specificity of PP2A/B55delta phosphatase, and in doing so, we will identify the phosphorylations that most require Greatwall-mediated protection from the phosphatase so as to ensure M phase entry and maintenance.